Further characterization of a neurotensin-degrading neutral metalloendopeptidase from rat brain

Abstract

A peptidase inactivating neurotensin at the Pro 10-Tyr 11 peptidyl bond, leading to the biologically inactive fragments neurotensin 1–10 and neurotensin 11–13 was purified from rat brain homogenate. The peptidase was characterized as a 70 kDa monomer and could be classified as a metaliopeptidase with respect to its sensitivity to o- phenanthroline, EDTA and divalent cations. The enzyme was also strongly inhibited by dithiothreitol but appeared totally insensitive to thiol-blocking agents, acidic and serine protease inhibitors. Experiments performed with a series of highly specific peptidase inhibitors clearly indicated that the peptidase was a novel enzyme distinct from previously purified cerebral peptidases. The enzyme displayed a rather high affinity for neurotensin ( Km = 2.3 it M). Studies on its specificity indicated that: (i) neurotensin 9–13 was the shortest neurotensin fragment with full inhibitory potency of [ 3H]neurotensin degradation. Shortening the C-terminal end of the neurotensin molecule progressively led to inactive analogs; (ii) the peptidase exhibited a strong stereospecificity towards the residues in positions 8, 9 and 11. By contrast, neither introduction of a steric hindrance in position 11 nor amidation of the C-terminal end of the neurotensin molecule affected the ability of the corresponding analog to inhibit [ 3H]neurotensin degradation; (iii) Pro-Phe was the most potent dipeptide to compete for [ 3H]neurotensin degradation; (iv) the peptidase could not be described as an exclusive “neurotensinase” activity since, in addition to the neurotensin natural analogs (neuromedin N and xenopsin), non related natural peptides such as angiotensins I and II, dynorphins 1–8 and 1–13, atriopeptin III and bradykinin potently inhibited [ 3H]neurotensin degradation. Most of these peptides behaved as substrates for the enzyme.